Nat Prod Sci.  2017 Mar;23(1):21-28. 10.20307/nps.2017.23.1.21.

Diarylbutane-type Lignans from Myristica fragrans (Nutmeg) show the Cytotoxicity against Breast Cancer Cells through Activation of AMP-activated Protein Kinase

Affiliations
  • 1College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea.
  • 2Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea. wkoh1@snu.ac.kr
  • 3Department of Pathology, College of Dentistry, Chosun University, Gwangju 501-759 Republic of Korea.

Abstract

In our program to search for new AMP-activated protein kinase (AMPK) activators from plants that exert potential anticancer property, we found that an EtOAc extract of Myristica fragrans (nutmeg) activated AMPK enzyme in human breast cancer MCF-7 cells. Two major diarylbutane-type lignans, macelignan and meso-dihydroguaiaretic acid (MDGA), were isolated as active principles from this extract. Treatment of breast cancer cells with two compounds induced cellular apoptosis, evidenced by cleavage of poly-(ADP-ribose) polymerase (PARP) and Ser 15 phosphorylation of p53. Moreover, macelignan and MDGA significantly inhibited the colony formation of MCF-7 breast cancer cells on soft agar. Intraperitoneal injection of macelignan and MDGA (20 mg/kg) suppressed the tumor growth of 4T1 mammary cancer cells. These results indicate that the chemopreventive effects of two major diarylbutane-type lignans from Myristica fragrans (nutmeg) may be associated with induction of apoptosis presumably through AMPK activation.

Keyword

Myristica fragrans; Diarylbutane lignan; MCF-7; AMP-activated protein kinase (AMPK)

MeSH Terms

Agar
AMP-Activated Protein Kinases*
Apoptosis
Breast Neoplasms*
Breast*
Humans
Injections, Intraperitoneal
Lignans*
MCF-7 Cells
Myristica fragrans*
Phosphorylation
AMP-Activated Protein Kinases
Agar
Lignans

Figure

  • Fig. 1 A representative HPLC profile of major compounds from the total EtOAc layer of Myristica fragrans with detections at 205 and 280 nm. Key to peak identity: (1) macelignan, (2) MDGA. Chromatographic method used: 0 - 40 min (50 - 70% MeOH), 40 - 52 min (70 - 100% MeOH), 52 - 60 min (100% MeOH).

  • Fig. 2 Phosphorylation of AMPK by macelignan and MDGA in MCF-7 cells. Cells were seeded using DMEM supplemented with 10% heat-inactivated fetal bovine serum without antibiotic and cultured for 24 hrs at 37℃ in humidified air condition containing 5% CO2. The cells were then starved with serum free DMEM media for 24 hrs and treated with 5 µg/ml of macelignan and MDGA for 10 min, 30 min, 1 hr, and 2 hrs (the left panels of A and B) or treated with 0, 1, 2.5, 5, and 10 µg/ml of macelignan and MDGA (the right panels of A and B), respectively. The cells were harvested with cold PBS and lysed with 1 × NP40 lysis buffer. Proteins in whole cell lysates were separated by SDS-PAGE and immunoblotted with antibodies against phospho-AMPK and total AMPK. AICAR, an AMPK activator, was used as a positive control in this experiment. Data are representative of three independent experiments that gave similar results.

  • Fig. 3 Inhibitory effects of macelignan and MDGA on colony formation of MCF-7cells. MCF-7 cells were subjected to soft agar assays in the presence of macelignan and MDGA. Cells (8 × 103/mL) were exposed to 1, 5, 10, and 20 µg/ml of macelignan and MDGA, respectively, in 1 ml of 0.3% basal medium Eagle (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 µg/ml gentamicin. The cultures were maintained at 37℃, in a 5% CO2 incubator for 10-14 days, and the cell colonies were scored using a microscope and the Image-Pro PLUS computer software program (Media Cybernetics, Silver Spring, MD).

  • Fig. 4 Effects of macelignan and MDGA on PARP cleavage and p53 phosphorylation on MCF-7 cells. Cells were cultured using DMEM supplemented with 10% heat-inactivated fetal bovine serum for 24 hrs at 37℃. The cells were then starved with serum free DMEM media for 24 hrs and treated with 5 µg/ml of macelignan and MDGA for 1, 3, 6, and 12 hrs (the upper panels of A and B) for cleaved PARP and treated with 0, 1, 2.5, 5, and 10 µg/ml of macelignan and MDGA for 30 min for phospho-P53 (the lower panels of A and B), respectively. AICAR was used as a positive control. Data are representative of three independent experiments that gave similar results.

  • Fig. 5 In vivo anti-cancer effect on 4T1 mammary cancer cells. Six-week-old female Balb/c mice were anesthesized with 50 mg/kg pentobarbital and the rudimentary mammary ducts was cleared. 4T1 mammary cancer cells were harvested by trypsinization and centrifuged and resuspended in DMEM at a density of 3 × 106/100 µl. 100 µl cell mixtures were injected into the cleared fat pad. Macelignan and MDGA (20 mg/kg) or an equal volume of the vehicle (n = 5 - 6 mice each group) were intraperitoneally injected by every two days for a total of 8 times. At the end of the treatment period, animals were sacrificed and solid tumors were excised for further studies. Body weights were recorded daily.


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